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Supplemental Data S1
Molecular Evidence
for Species-Level Distinctions
in Clouded Leopards
Valerie A. Buckley-Beason, Warren E. Johnson,
Willliam G. Nash, Roscoe Stanyon, Joan C. Menninger,
Carlos A. Driscoll, JoGayle Howard, Mitch Bush,
John E. Page, Melody E. Roelke, Gary Stone,
Paolo P. Martelli, Ci Wen, Lin Ling,
Ratna K. Duraisingam, Phan V. Lam,
and Stephen J. O’Brien
Supplemental Experimental Procedures
Biological Samples
Biological samples were obtained from captive animals of known
geographic origin (n = 42), from captive born animals from parents
of known geographic origin (n = 38), and from museum specimens
(n = 29) (Figure 1). For outgroup comparison, fifteen broadly distrib-
uted individuals of known geographic origin from species of Pan-
thera (P. tigris,P. pardus,P. uncia, and P. onca) were selected along
with ten individuals from each of six well-studied lion (P. leo) popula-
tions, along with two domestic cats (Felis catus) (Tables S1 and S2).
DNA was extracted from white blood cells, skin fibroblast cells, or
tissues according to a phenol-chloroform protocol [S1]. Museum
samples of hide, hair, or bone were extracted with a modified sil-
ica-based, guanidium extraction method [S2–S4]. Cell lines for
DNA extraction and cytogenetic analyses were established accord-
ing to standard methods [S5].
Mitochondrial and Nuclear Gene Amplification
and Phylogenetic Analysis
Sequence variation among clouded leopards and outgroup species
was assessed for portions of four mitochondrial (mtDNA) gene seg-
ments, one autosomal gene segment (HK1), and four X-linked gene
segments (ATP7A,BGN,IDS and PLP). Gene fragments were ampli-
fied by PCR of genomic DNA in 96-well-plate format with 76 clouded
leopards and two samples from each Panthera genus species and
two domestic cats.
For the mtDNA genes, 139 bp of ATPase-8 (ATP-8) was amplified
with primers ATP-8[F] 50-ACAACTAGATACATCCACCTGA-30and
[R] 50-GGCGAATAGATTTTCGTTCA-30, 223 bp of Sodium Dehydro-
genase-5 (ND5) with primers (F) 50-GTGCAACTCCAAATAAAAG-30
and (R) 50-TAAACAGTTGGAACAGGTT-30, 219 bp of Cytochrome-
b(Cyt-b) with (F) 50-ATGACCAACATTCGAAAATC-30and (R) 50-
TGTATAGGCAGATAAAGAATATGGA-30, and 190 bp of the Control
Region (CR1) with (F) 50-TCAAAGCTTACACCAGTCTTGTAAACC-30
and [(R) TAACTGCAGAAGGCTAGGACCAAACCT-30]. ATP-8 and
Cyt-b primers sets were designed from consensus felid sequences
to reduce the probability of amplifying human genomic DNA result-
ing from handling. Nuclear gene fragments were amplified with
primers from a previous study [S6, S7].
PCR reactions were set in 25 ml volume reactions with 2.5 mlof
10 ng/ul DNA, 2.5 ml103Perkin Elmer PCR buffer, 2 mM MgCl
2
,
250 mM each of the four deoxyribonucleoside 50-triphosphates
(dATP, dCTP, dGTP, and dTTP). (Pharmacia), 2.5 units of AmpliTaq
Gold DNA polymerase (Perkin-Elmer), and 20 mM of each forward
and reverse primer (Life Technologies and PE Applied Biosystems).
Amplification consisted of an initial denaturing of 10 min at 95C for
12 cycles with a 2C decreasing annealing temperature (three cycles
at 62C, 60C, 58C, and 56C), continued with 30 cycles with an
annealing temperature of 54C, and ended with a 7 min extension
at 72C. Both strands of PRC products were sequenced.
Resulting raw sequences were cleaned with SEQUENCHER (Mac
Version 4.1.2) and were verified with BLAST genomic searches [S8]
before being exported to PAUP (Version 4.0b10) [S9] for phyloge-
netic analysis. Phylogenetic analyses of sequence variation were
conducted for each gene segment and for 40 individuals with a con-
catenated sequence of 3,859 bp. Phylogenetic relationships among
the sequences were reconstructed with minimum-evolution (ME),
maximum-parsimony (MP), and maximum-likelihood (ML) ap-
proaches [S9, S10]. Support for the nodes in each analysis was
assessed by 1000 bootstrap replicates. Each phylogenetic tree anal-
ysis was rooted with the domestic-cat sequence. Support for the no-
des in each analysis was assessed by 1000 bootstrap replicates.
The approximate age of separation of modern clouded leopards
was estimated with LINTREE [S11]. LINTREE tests the molecular
clock on a given topology of a phylogenetic tree and makes linear-
ized trees while re-estimating branch lengths under the assumption
of a constant rate of evolution [S11]. A phylogenetic tree was con-
structed with the NJ method [S10] and Kimura 2 parameter distance
for all the mtDNA and nuclear sequences combined (3.9 Kb). Both
the two-cluster and branch-length tests implemented in LINTREE
were applied [S11]. The molecular-clock tests implemented in LIN-
TREE [S11] showed that the set of clouded leopard sequences did
not deviate significantly from the rate-constancy test (p > 0.05).
Microsatellite Amplification and Phylogenetic Analysis
Patterns of microsatellite size variation were assessed in clouded
leopards and outgroup species with 51dinucleotide microsatellite
primers randomly chosen from a panel of more than 300 polymor-
phic loci characterized in the domestic cat [S12]. These microsatel-
lites are located on 17 of the 18 felid autosomes and are all greater
than five centimorgans (cM) apart except for markers FCA 5, 26,
75, 82, 85, 161, 171, 224, and 249 (Table 2). PCR amplification of
the 51 individual dinucleotide microsatellite loci was performed in
a10ml final reaction volume. All loci were amplified with 10 ng/ml
DNA with previously described microsatellites and methods [S12].
Final base callings were performed with the ABI Genotyper package
(version 2.1), and allele sizes were estimated by the Local Southern
method [S13].
The relationships among clouded leopards and the out-group
species were assessed with seven different algorithms based pri-
marily on two different approaches. Three methods, delta mu
squared (jdmj
2
), average square distance (ASD), and Dsw, use differ-
ences in allele sizes as an indication of phylogenetic distance, and
Rst measures the inter-population diversity of alleles [S14, S15].
The remaining three algorithms, proportion of shared alleles (Dps),
Nei’s standard distance (Gst), and kinship coefficient distance
(Dkf), rely on the degree of similarity among microsatellite size dis-
tribution and the amount of overlap between allele frequencies to
estimate relatedness. Composite genotypes were compared via
genetic distances obtained with MICROSAT2 [S16], with 100 boot-
strap resampling iterations of 100 [S17]. MICROSAT2 was also
utilized to estimate genetic variation. Neighbor joining (NJ) and con-
sensus programs within the Phylogeny Inference Package (PHYLIP,
Version 3.5) [S18] were utilized to produce phylogenetic trees based
upon these distances. Tree files produced by PHYLIP were visual-
ized with TreeView (Version 1.6.1).
Seven microsatellite genetic-distance algorithms were used to ex-
amine the relationships among the clouded leopard subspecies.
Analyses based on allelic size differences (ASD, jdmj
2
, Dsw) united
all clouded leopards into a monophyletic group versus other felid
species, with a dichotomous separation of subspecies N. n. diardi
separated from N. n. nebulosa individuals (Figure 1D in the main
text). The remaining four algorithms (Dps, Dkf, Fst, and Gst), based
upon microsatellite allelic overlap, differed in that the individuals
from N. n. diardi formed a distinct group more closely aligned to
out-group species than to N. n. nebulosa (Figure 1D in the main
text and Figures S5–S8). The large difference in the allele-overlap
methods reflects the large number of nonoverlapping alleles in 20
of 51 loci between the two clouded leopard subspecies; this is twice
Figure S1. Variable Site Alignment across 771 bp of mtDNA
Clouded leopards were depicted as mtDNA haplotypes and aligned with a single haplotype from each species from the Panthera lineage and a domestic cat. Missing or ambiguous data are identified with an
asterisk, and gaps in the sequence are designated with an dash. Numbers represent the alignment position based upon the sequences placed in alphabetical order (APT-8, CR, Cyto-b, ND5, ATP-7A, BGN,
HK1, IDS, and PLP) with mitochondrial genes first. The number next to the haplotypes represents the number of clouded leopard individuals that shared that same haplotype. Total nucleotides assessed com-
prised 3.8 kb. Subspecies abbreviations are as in Table 1.
S2
Figure S2. Variable Site Alignment across 3.1 kb of Nuclear Sequence
See the legend for Figure 1.
S3
the number of nonoverlapping alleles found between any two
species within Panthera (Figure S9).
A Bayesian clustering method implemented in STRUCTURE [S19]
was used to infer population structure based upon multilocus geno-
type data. We calculated the probability of individual assignments to
population clusters (K). A series of tests were conducted with differ-
ent numbers of population clusters in order to guide an empirical
estimate of the number of identifiable populations; for these tests,
we assumed an admixture model with correlated allele frequencies
and set burnin at 50,000 and replication at 10,000, respectively.
Each test yielded a log likelihood value of the data (Ln probability),
the highest of which would indicate which test was closest to the
actual number of genetically distinct populations. The statistical
significance of Rst values was derived from AGARst [S20] and
GENEPOP [S21].
Chromosome Sorting and Hybridization
Karyotype analysis of nine individuals from N. n. nebulosa and three
from N. n. diardi was performed with G banding. Adobe Photoshop
was used for pairing the individual’s metaphase chromosomes with
their homologs and arranging them in the standard Felidae layout for
visual comparison among clouded leopard individuals [S22, S23].
Mainland clouded leopard chromosomes were sorted by a dual-
laser cell sorter (FACSDiVa) that allows a bivariate analysis of chro-
mosomes by size and base-pair composition [S24]. We sorted 500
chromosomes from various peaks containing different clouded
leopard chromosomes directly into PCR tubes containing 30 mlof
distilled water. The chromosomal DNA was then amplified by degen-
erate oligonucleotide primed PCR (DOP-PCR) with the 6MW primer
(50-CCGACTCGAGNNNNNNATGTGG-30). The probes were labeled
with biotin dUTP with the same primer [S25, S26].
Common FISH procedures were followed for in situ hybridization
and probe detection [S27]. About 300 ng of each PCR product per
probe, together with 10 mg of clouded leopard Cot-1 DNA, was pre-
cipitated and then dissolved in 14 ml hybridization buffer. After hy-
bridization and washing of the slides, biotinylated DNA probes
were detected with avidin coupled with fluorescein isothiocyanate
(FITC, Vector).
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Figure S3. Phylogenetic Tree of All mtDNA
Phylogenetic tree among DNA sequence genotypes of Neofelis and Panthera lineages. All mtDNA gene segments (771 bp) are included.
S4
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Figure S4. Phylogenetic Tree of All Nuclear-Sequence Data
All nuclear gene segments (3.1 kb) are included.
S5
Figure S5. Phylogenetic Tree Based on Fst Microsatellite Distance Measure
Figures S5–S8 show phylograms constructed by various algorithms (Fst, Gst, Dkf, & jdmj
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51 microsatellites. Values depict bootstrap support (of 100 iterations).
S6
Figure S6. Phylogenetic Tree Based on Gst Microsatellite Distance Measure
S7
Figure S7. Phylogenetic Tree Based on Dkf Microsatellite Distance Measure
S8
Figure S8. Phylogenetic Tree Based on jdmj
2
Microsatellite Distance Measures
S9
Figure S9. Difference Plots of Minimum Number of Nucleotides Separating Overlapping Microsatellite Allele Sizes
Distinction of nonoverlapping microsatellite locus allele sizes between N. n. nebulosa subspecies (A) and between individual Pathera species
(B–F).
S10
Figure S10. FISH Results of F Chromosomes
of N. n. nebulosa and N. n. diardi
(A) Fluorescent in situ hybridization (FISH) of
DOP-PCR labeled E group chromosome to
N. n. nebulosa illuminates index chromosome
(p) and telomeres of several other chromo-
somes (arrow).
(B) FISH of DOP-PCR labeled E group chro-
mosome to N. n. diardi illuminates index
chromosome (p) but not telomeres of other
chromosomes.
S11